Frequent Detection and Genetic Diversity of Human Bocavirus in Urban Sewage Samples

The prevalence and genetic diversity of human bocaviruses (HBoVs) in sewage water samples are largely unknown. In this study, 134 raw sewage samples from 25 wastewater treatment plants (WTPs) in Italy were analyzed by nested PCR and sequencing using species-specific primer pairs and broad-range primer pairs targeting the capsid proteins VP1/VP2. A large number of samples (106, 79.1 %) were positive for HBoV. Out of these, 49 were classified as HBoV species 2, and 27 as species 3. For the remaining 30 samples, sequencing results showed mixed electropherograms. By cloning PCR amplicons and sequencing, we confirmed the copresence of species 2 and 3 in 29 samples and species 2 and 4 in only one sample. A real-time PCR assay was also performed, using a newly designed TaqMan assay, for quantification of HBoVs in sewage water samples. Viral load quantification ranged from 5.51E+03 to 1.84E+05 GC/L (mean value 4.70E+04 GC/L) for bocavirus 2 and from 1.89E+03 to 1.02E+05 GC/L (mean value 2.27E+04 GC/L) for bocavirus 3. The wide distribution of HBoV in sewages suggests that this virus is common in the population, and the most prevalent are the species 2 and 3. HBoV-4 was also found, representing the first detection of this species in Italy. Although there is no indication of waterborne transmission for HBoV, the significant presence in sewage waters suggests that HBoV may spread to other water environments, and therefore, a potential role of water in the HBoV transmission should not be neglected.     

Human Bocavirus: Detection,Quantification and Molecular Characterization in Sewage and Surface Waters in Uruguay

Human bocavirus (HBoV) infections are related to respiratory and gastroenteric diseases. The aim of this study was to investigate the presence of HBoV in both sewage and surface waters in Uruguay. Sixty-eight sewage samples from the cities of Salto, Paysandú, Bella Unión, Fray Bentos, Treinta y Tres and Melo and 36 surface water samples from the cities of Salto, Florida and Santa Lucía were studied. HBoV was screened by multiplex qPCR for the detection of the four subtypes, followed by monoplex qPCRs for the independent quantification of each subtype. A qualitative PCR followed by DNA sequencing and phylogenetic analysis was carried out for molecular characterization of HBoV strains. HBoV was present in a high frequency (69%) in sewage and only one positive sample (3%) was found in surface water. Concerning sewage samples, HBoV1 was detected in 11 (23%) out of the 47 positives samples, with a mean concentration of 8.2 × 10⁴ genomic copies/Liter (gc/L), HBoV3 was detected in 35 (74%) of the positive samples with a mean concentration of 4.1 × 10⁶ gc/L and subtypes 2 and/or 4 were detected in 39 (83%) of the positive samples with a mean concentration of 7.8 × 10⁶ gc/L. After the phylogenetic analysis performed by a Bayesian approach, the four HBoV subtypes were confirmed. This is the first study determining a high frequency of HBoV and the presence of the four HBoV subtypes in aquatic matrices in Latin America, mainly in sewage. Although HBoV was scarcely detected in surface water, a waterborne transmission is likely to occur if people enter in contact with polluted surface waters for recreational activities such as fishing or swimming since an elevated frequency of HBoV was detected in raw sewage which is usually directly discharged into surface waters.     

Relative Abundance of Human Bocaviruses in Urban Sewage in Greater Cairo,Egypt

Human bocavirus (HBoV) is predominantly found in the respiratory tract infections and in the stool of patients with gastroenteritis symptoms. However, data on the prevalence of HBoV genotypes in environmental samples are limited. Here we addressed the prevalence of HBoV in sewage collected from three different wastewater treatment plants in Egypt. HBoV-1, HBoV-2, and HBoV-3 were detected, whereas none of the samples were positive for HBoV-4. The median concentration of HBoV in influent samples was 8.5 × 10³ GC/l for HBoV-1, 3.0 × 10⁴ GC/l for HBoV-2, and 2.5 × 10⁴ GC/l for HBoV-3. The concentration was reduced but not completely removed in the effluent samples. The median concentration in the outlet samples was 2.9 × 10³ GC/l for HBoV-1, 4.1 × 10³ GC/l for HBoV-2, and 2.1 × 10³ GC/l for HBoV-3. Moreover, no seasonality pattern of HBoVs was found. The high incidence of HBoV in sewage samples provided an evidence of its circulation in the local population. Although the role of HBoV in respiratory or gastro-intestinal infections still remains to be fully elucidated, the risk of infection via contaminated water should be taken into consideration.     

Rotavirus Surveillance in Tap Water,Recycled Water,and Sewage Sludge in Thailand: A Longitudinal Study, 2007–2018

The objective of this study was to describe the epidemiological and molecular surveillance of rotaviruses in tap water, recycled water, and sewage sludge in Thailand from 2007 to 2018. Three hundred and seventy tap water, 202 recycled water, and 72 sewage sludge samples were collected and processed to detect the rotavirus VP7 gene using RT-nested PCR. Rotavirus G genotypes were identified by DNA sequencing and phylogenetic analysis. The frequency of rotavirus detection was 0.54% of the tap water samples, 30.2% of the recycled water samples, and 50.0% of the sewage sludge samples. During the 12-year surveillance, G1 was prevalent most years and constantly predominant in recycled water and sewage sludge. G2 was identified in a tap water sample and in recycled water samples. G3 and G9 were observed in both recycled water and sewage sludge samples. The uncommon G6 rotavirus strain was identified in one recycled water sample. The rotavirus VP4 gene was detected in rotavirus strains with an identified G genotype using RT-multiplex nested PCR. The unusual P[6] genotype was the most frequently detected, followed by mixed P[6]/[4] and P[4] genotypes. Phylogenetic analysis of both G and P genotypes showed a close genetic relationship with sequences of human rotavirus strains. The high nucleotide identity of the rotavirus strains found in this study to human rotavirus strains suggests that the rotaviruses are derived from human source. These results represent useful epidemiological and molecular information for evaluating rotavirus distribution in water for consumption and irrigation, and in biosolids for agricultural application.

     

Clinical and Environmental Surveillance of Rotavirus Common Genotypes Showed High Prevalence of Common P Genotypes in Egypt

The objective of this study was to compare the prevalence of human rotavirus group A common G and P genotypes in human Egyptian stool specimens and raw sewage samples to determine the most common genotypes for future vaccine development. From 1026 stool specimens of children with acute diarrhea and using nested RT-PCR, 250 samples (24.37%) were positive for human rotavirus group A. Using multiplex RT-PCR, rotavirus common P and G genotypes were detected as 89.20% and 46.40% of the positive clinical specimens respectively. This low percentage of common G genotypes frequency may affect the efficiency of the available live attenuated oral rotavirus vaccines [Rotarix® (human rotavirus G1P[8]) and RotaTeq® (reassortant bovine–human rotavirus G1-4P[5] and G6P[8])], however the percentage of clinical specimens which were negative for common G genotypes but positive for P[8] genotype was 12.00%. From 24 positive raw sewage samples for rotavirus group A VP6 collected from Zenin and El-Gabal El-Asfar wastewater treatment plants (WWTPs), 21 samples (87.50%) were typeable for common P genotypes while 13 samples (54.17%) were typeable for common G genotypes. Phylogenetic analysis of a VP8 partial gene of 45 P-typeable clinical isolates and 20 P-typeable raw sewage samples showed high similarity to reference strains and the majority of mutations were silent and showed lower to non-significant similarity with the two vaccine strains. This finding is useful for determining the most common antigens required for future vaccine development.

     

青岛近海沉积物PCBs的水平与垂直分布及贝类污染

利用13C同位素内标法及气相色谱-质谱方法对青岛胶州湾内外8个表层沉积物,1个沉积物柱状岩心和2个贝类样品中包括14种共平面PCBs在内的50种PCBs同系物含量进行了测定,在所有样品中均检测出了PCBs化合物.讨论了PCBs的分布特征.表层样品以胶州湾东岸的含量最高,从北岸、西岸、胶州湾口及胶州湾外PCBs含量逐渐降低.表层沉积物中总PCBs含量在0.65～32.9ng/g dw. PCBs总含量落在国内外海洋近岸表层沉积物含量范围的中值区.表明污染来源有城市污水排放及大气来源2种,而不属于工业污染类型.多氯联苯主要以低氯取代PCBs为主,大部分高氯取代PCBs的含量低于检出限.柱状样中共平面PCBs的含量及毒性当量浓度总的垂直变化趋势是从1951年到现在逐渐减少.贝类样品的总PCBs含量为4.9～8.4ng/g dw,属低污染水平.     

Environmental Surveillance for Noroviruses in Selected South African Wastewaters 2015–2016: Emergence of the Novel GII.17

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2–3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 10² to 3.41 × 10⁶ genome copies/litre for GI and 5.00 × 10³ to 1.31 × 10⁶ genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.     

Detection and Characterization of Hepatitis A Virus and Norovirus in Mussels from Galicia (NW Spain)

Shellfish are recognized as a potential vehicle of viral disease and despite the control measures for shellfish safety there is periodic emergence of viral outbreaks associated with shellfish consumption. In this study a total of 81 mussel samples from Ría do Burgo, A Coruña (NW Spain) were analysed. Samples were collected in seven different harvesting areas with the aim to establish a correlation between the prevalence of norovirus (NoV) and hepatitis A virus (HAV) in mussel samples and the water quality. In addition, the genogroup of the detected HAV and NoV strains was also determined. The HAV presence was detected in 18.5 % of the samples. Contamination levels for this virus ranged from 1.1 × 10² to 4.1 × 10⁶ RNA copies/g digestive tissue. NoV were detected in 49.4 % of the cases reaching contamination levels from 5.9 × 10³ to 1.6 × 10⁹ RNA copies/g digestive tissue for NoV GI and from 6.1 × 10³ to 5.4 × 10⁶ RNA copies/g digestive tissue for NoV GII. The χ²-test showed no statistical correlation between the number of positive samples and the classification of molluscan harvesting area based on the E. coli number. All the detected HAV strains belong to genogroup IB. NoV strains were assigned to genotype I.4, II.4 and II.6.     

Isolation and Identification of Enteroviruses from Sewage and Sewage-Contaminated Water in Lagos,Nigeria

Studies have confirmed silent circulation of enteroviruses in the environment even in the absence of associated clinical conditions in the community. In this light, 26 samples of sewage and sewage-contaminated water serving selected high-risk communities in Lagos Nigeria were examined between June and September 2010. To concentrate virus particles in the sample, 480 μL of each sample was centrifuged at 3,000 rpm for 1 h at 4 °C. Subsequently, pellets were pooled, chloroform treated and further centrifuged at 1,500 rpm for 20 min at 4 °C. The water phase (concentrate) was then collected and stored at ?20 °C. The concentrates were subsequently inoculated into RD and L20B cell lines. Recovered isolates were identified by real-time RT-PCR (rRT-PCR), serotyping, VP1 amplification, sequencing and phylogenetic analysis. Overall, 9 (34.6 %) of the samples showed characteristic enterovirus cytopathic effect in RD cell line and were subsequently confirmed by pan-enterovirus rRT-PCR. The isolates were further identified by serotyping to include three E7, one E11 and one E13 isolates whilst four isolates were untypable. Further characterisation by VP1 sequencing confirmed the results of serotyping and rRT-PCR for all but isolate E13. Also, the four previously untypable isolates were identified to include two E19, one E20 and one E7 by VP1 sequencing. Results of the study confirmed circulation of Sub-Saharan Africa-specific enterovirus clades in the region, provide information on their molecular epidemiology and emphasise the need to combine methods of identification to enhance enterovirus surveillance.     

A Phororhacoid bird from the Eocene of Africa

The bird fossil record is globally scarce in Africa. The early Tertiary evolution of terrestrial birds is virtually unknown in that continent. Here, we report on a femur of a large terrestrial new genus discovered from the early or early middle Eocene (between ～52 and 46?Ma) of south-western Algeria. This femur shows all the morphological features of the Phororhacoidea, the so-called Terror Birds. Most of the phororhacoids were indeed large, or even gigantic, flightless predators or scavengers with no close modern analogs. It is likely that this extinct group originated in South America, where they are known from the late Paleocene to the late Pleistocene (～59 to 0.01?Ma). The presence of a phororhacoid bird in Africa cannot be explained by a vicariant mechanism because these birds first appeared in South America well after the onset of the mid-Cretaceous Gondwana break up (～100?million years old). Here, we propose two hypotheses to account for this occurrence, either an early dispersal of small members of this group, which were still able of a limited flight, or a transoceanic migration of flightless birds from South America to Africa during the Paleocene or earliest Eocene. Paleogeographic reconstructions of the South Atlantic Ocean suggest the existence of several islands of considerable size between South America and Africa during the early Tertiary, which could have helped a transatlantic dispersal of phororhacoids.     

GIV Noroviruses in Wastewaters and in Stool Specimens from Hospitalized Patients

Noroviruses (NoVs) are important human pathogens associated with foodborne and waterborne gastroenteritis. These viruses are genetically highly heterogeneous, with more than forty genotypes within three genogroups (GI, GII, and GIV) identified in humans. However, the vast majority of human infections are associated with variants of a unique genotype, GII.4. Aside from these NoV strains of epidemiological relevance, NoV strains of genogroup GIV (Alphatron-like) are reported in a sporadic fashion and their overall prevalence in the community is unknown and this likely reflects the lack of specific diagnostic tools. We analyzed raw sewages collected from 32 wastewater treatment plants distributed throughout Italy (307 samples) and stool specimens collected from hospitalized patients with clinical signs of diarrhea of unknown etiology (285 samples). By using specific qualitative and quantitative RT-PCR assays, 21.8 % of the sewage samples and 3.2 % of the stool specimens tested positive for GIV NoVs. The number of genome copies in fecal samples ranged from 5.08 × 10⁴ to 1.73× 10⁶/g of feces. Sequence analysis showed limited genetic variability in human GIV viruses. The presence of GIV NoV both in sewage and in clinical samples confirms that not only GI and GII NoVs but also GIV strains are circulating in humans. Monitoring of GIV NoV is recommended in order to understand the dynamics of circulation in human populations, environmental contamination, and potential health risks.     

Carbon footprint analysis for increasing water supply and sanitation in South Africa: a case study

This study investigates the environmental burdens due to the provision of potable water and sanitation in the eThekwini Municipality (Durban), South Africa. This was achieved by employing LCA studies for the individual parts of the urban water system (impoundment, water treatment, distribution, collection, sewage treatment and water recycling). Based on the results of the individual LCAs a base case was constructed.For the provision of potable water and sanitation to new customers, which have not been previously served, two different scenarios (200 000 new customers in an urban environment with waterborne sewage and in a peri-urban environment with on-site sanitation) and three different options (maximising use of existing assets, recycling water and building new infrastructure) were considered and analysed. With regard to the impact scores calculated for both scenarios (urban and peri-urban), the recycling of water is followed by maximising the use of existing assets as the most environmentally friendly options. The construction of new infrastructure carries a higher environmental burden and the use of bottled water for drinking (an additional scenario) carries the highest environmental burden.     

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 10⁶) > treated sewage (2.9 × 10⁵) > river waters (6.1 × 10²) > estuarine waters (4.8 × 10²) > bathing waters (8.5 × 10¹) > groundwater (5.9 × 10¹). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

     

Detection of Enteroviruses in Influent and Effluent Flow Samples from Wastewater Treatment Plants in Italy

This study evaluated the presence and seasonal distribution of polio and other enteroviruses in four wastewater treatment plants in three cities in Italy, using different treatment systems. Detection of enteroviruses was carried out by virus isolation in cell cultures after concentration of water samples collected at both inlet and outlet of the treatment plants, following the methods described in the WHO guidelines. Viral serotypes isolated before and after water treatment were compared. Forty-eight non-polio enteroviruses were isolated from 312 samples collected at the inlet of the four wastewater treatment plants, 35 of which were Coxsackievirus type B (72.9 %) and 13 Echovirus (27.1 %). After treatment, 2 CVB3, 1 CVB5, and 1 Echo 6 were isolated. CVB3 and Echo 6 serotypes were also detected in samples collected at the inlet of the TP, in the same month and year. The high rate of detection of infectious enteroviruses in inlet sewage samples (30.1 %) indicates wide diffusion of these viruses in the populations linked to the collectors. The incomplete removal of infectious viruses following sewage treatment highlights possible risks for public health relate to treated waters discharge into the environment.     

中国城市污泥中汞含量的时空分布特征

为全面了解中国城市污水处理厂脱水污泥中汞的赋存状况,从全国40个城市污水处理厂采集315个污泥样品,使用DMA-80直接测汞仪测定了样品中汞的含量.结果表明,全国城市污泥样品中的汞含量范围为0.45~15.42 mg·kg~(-1),汞含量符合对数正态分布,几何平均值为(2.19±3.16)mg·kg~(-1).所有污泥样品中的汞含量均满足《城镇污水处理厂污泥处置混合填埋用泥质》(GB/T 23485-2009)规定的污泥填埋标准;97.8%的城市污泥汞含量均满足土地改良用泥质标准(中性、碱性土壤);86.7%的城市污泥汞含量满足土地改良用泥质标准(酸性土壤).不同城市的污泥样品汞含量存在较大差异(变异系数105%),同一个污水处理厂污泥汞含量也不稳定(7d变异系数0.6%~53.6%).污泥中汞含量中位数呈现华北地区东北地区西北地区西南地区华东地区华中地区华南地区的空间分布趋势.以相应城市的土壤背景值为参比,利用地累积指数法,对于城市污泥中的汞污染现状进行了评价,发现汞重度污染以上的省份比例达到60%以上.与历史数据对比发现,全国城市污泥中汞含量呈现先升高后降低的趋势,峰值出现于2000~2009年.研究结果为城市污水和污泥汞污染的防治工作提供了数据支持.     

Prevalence of Rotaviruses Groups A and C in Egyptian Children and Aquatic Environment

The objective of this study is to compare the prevalence of rotaviruses groups A and C in Egyptian children and aquatic environment. From 110 stool specimens of children with acute diarrhea and using RT-PCR, 35 samples (31.8 %) were positive for human rotavirus group A and 15 samples (13.6 %) were positive for human rotavirus group C. From 96 samples collected from Zenin wastewater treatment plant over a 2-year period (November 2009–October 2011) and using RT-PCR, rotavirus group A was detected in (4/24) 16.7 %, (5/24) 20.8 %, (4/24) 16.7 %, and (4/24) 16.7 %, while rotavirus group C was detected in (2/24) 8.3 %, (3/24) 12.5 %, (3/24) 12.5 %, and (0/24) 0 % in raw sewage, after primary sedimentation, after secondary sedimentation, and after final chlorination, respectively. Moreover, from 96 samples collected from El-Giza water treatment plant over a 2-year period (November 2009–October 2011), rotavirus group A was detected in (7/24) 29.2 %, (6/24) 25 %, (5/24) 20.8 %, and (3/24) 12.5 %, while rotavirus group C was detected in (3/24) 12.5 %, (1/24) 4.2 %, (1/24) 4.2 %, and (0/24) 0 % in raw Nile water, after sedimentation, after sand filtration, and after final chlorination, respectively. Using SYBR Green real-time RT-PCR, the number of human rotavirus group A genome or infectious units was higher than rotavirus group C. VP6 sequence analysis of the RT-PCR positive rotavirus group C samples revealed that four clinical specimens and three environmental samples showed similar sequences clustered with Moduganari/Human Nigerian strain AF 325806 with 98 % homology, and two clinical specimens and one environmental sample showed similar sequences clustered with Dhaka CB/Human Bangladesh strain AY 754826 with 97 % homology.